LAB 2 WRITE-UP

Thermal Cycler Engineering

Our re-design is based upon the Open PCR system originally designed by Josh Perfetto and Tito Jankowski.

System Design
To begin with we will change the frame from a wooden construction to recycled aluminum. This will maintain the light-weight portability of the machine, but will greatly reduce the fire hazard of heating a wooden box to the required temperatures. We will also add an additional row and column to the heating block to increase the product output without too much change to the overall size or cost of the machine.
We will also add two carrying handles to either side of the machine. Because the machine is somewhat awkward to carry and hold the handles will greatly reduce the chances of dropping if they are properly used.

Key Features

Instructions

Protocols

Materials

Supplied in PCR Kit

Amount

PCR Machine

1

Fluorometer Box

1

Camera Stand

1

Glass Slides

4

Calf Thymus

0.5 mL

Calibration DNA

0.5 mL

GoTaq Polymerase and Buffer Solution

10 mL

Small Test Tubes

25

Large Test Tubes

25

SYBR Green (diluted)

1.5 mL

Supplied by User

Amount

Smartphone Camera

1

Laptop

1

ImageJ Download

1

Power Supply

1

DNA Sample

At least 3 samples

PCR Protocol

1. Using a micro-pipette, transfer 1.0-1.5 micro liters of desired DNA sample into at least three test tubes.

2. Micro-pipette approximately 3.0 micro liters of reagent solution, including forward primer, reverse primer, GoTaq polymerase and buffer solution, into each of the test tubes with the DNA.

3. Invert tubes, then turn upright to mix solution.

4. Plug in OpenPCR Machine and turn it on.

3. Open lid and place tubes into holder in PCR machine (can fit up to 25 tubes). Close the lid.

6. After cycles are complete, open the lid and remove tubes for signal reading (see DNA Measurement Protocol below).

DNA Measurement Protocol

1. Remove lid from black box and invert the box, open the front flap.

2. Place one glass slide into the holder.

3. Depending on the amount of samples being used, take a clean pipette for each sample and label it so that it is only used for that sample of DNA. For example, label a pipette for SYBR green with a horizontal line on the pipette bulb, and label a pipette for diluted water with a vertical line on the bulb. Do NOT use pipettes for any other solution than what they are labelled for.

4. Using the designated pipette, squeeze two drops of SYBR green dye inside the round glass windows on slide, preferably the second dot of the second row.

5. Begin by adding a sample of diluted water to the SYBR green dye, and using the appropriate labelled pipette, add two drops in the same spot as the SYBR green dye.

6. Align the sample so the blue LED shines directly though it focusing the light on the other side.

7. Move holder apparatus inside the box so that no light reaches it.

8. Place smartphone with the flash off onto holder and direct camera lens at the slide apparatus.

9. Take photo with smartphone.

10. E-mail the taken image to computer.

11. On the computer open the image file using ImageJ (program can be downloaded from the Internet.)

13. Make sure the following boxes are checked, and no other boxes other than the ones listed:
-Area
-Mean Grey Value
-Integrated Density

14. From the download folder drag the image file into ImageJ.

15. When the image is opened in ImageJ from the top drop down menu select "Image" and then "Color" and then "Split Channels."

16. Close out the red and blue channel, only use the green channel

17. With the Oval tool select only the entire drop of liquid, avoiding the background but encompassing every bit of the droplet.

18. After the droplet part of the image is selected, go to "Analyze" then "Measure" or press Ctrl + M.

19. Now move the circle to the background of the image by clicking and dragging on the circle, be careful not to change its size.

20. Save the results as an excel spreadsheet (set by default).

21. Once the Image has been processed, using a clean pipette, remove the sample of diluted water and SYBR green dye from the slide.

22. Repeat steps 4-21 using included calf thymus DNA as a control, and move back on the glass slide two rows to eliminate contamination.

23. Once the control has been analyzed, repeat step 4-21 using the sample DNA, ensuring to move back two rows on the glass slide each time a new sample is tested.

24. Once there are no more rows on the glass slide, carefully discard the used slide and begin using a new one.

Research and Development

Background on Disease Markers

The SNP chosen for the lab is linked with Alzheimer's disease. Alzheimer's disease is the most common form of dementia. It has no cure and worsens as it progresses. Dementia is a loss of brain function and Alzheime's disease affects memory, thinking, and behavior.The associated SNP's for an increase in risk of Alzheimer's disease is rs1050283, which is located on the 12 chromosome.Information avaliable from the NCBI website:
http://www.ncbi.nlm.nih.gov/snp?term=rs1050283

Primer Design

The sequence that is connected with an increase risk of Alzheimer's disease is GGCTGGGCOCGGACATGGAGGACGTG[C/T]GCGGCCGCCTGGTGCAGTACCGCGG. The increase risk comes from the base change C to a T.
The reverse primer would be GGAGGACGTG[T]GCGGCCGCCT and the forward primer would be CCTCCTGCAG[A]CGCCGGCGGA.
A diseased allele will produce a PCR product because the gene associated with an increase risk that is trying to be tested for will have a T base in place of the normal C base. If the gene has this change in bases then the primers will be able to attach to the strands of DNA because the primers are designed to only attach to that specific sequence.